Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases

ABSTRACT

A quantitative method for performing an automated diagnostic assay, comprising: incubating a capture reagent with a streptavidin-coated medium to form a solid phase complex; washing the solid phase complex to remove excess capture reagent; incubating the solid phase complex with a serum sample to form an immune complex; washing the immune complex to remove any unbound sample; incubating the immune complex with a conjugate to create an immune-conjugate complex; washing the immune-conjugate complex to remove any unbound conjugate; introducing a substrate capable of generating a quantifiable response; and calibrating the response generated from introducing the substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/215,720, filed Mar. 17, 2014, which is related to, and claimspriority to, U.S. Provisional Patent Application Ser. Nos. 61/791,295and 61/791,879, each of which were filed on Mar. 15, 2013, the completeand entire disclosures of which are hereby expressly incorporated byreference herein.

TECHNICAL FIELD

The present teachings are related to a system and process for performingdiagnostic assays, and more particularly to an automated immunoanalyzersystem and process for performing diagnostic assays for allergies andautoimmune diseases.

BACKGROUND OF THE DISCLOSURE

The statements in this section merely provide background informationrelated to the present disclosure and should not be construed asconstituting prior art.

During an automated immunochemistry analysis, analyte molecules in apatient's biological sample (e.g., serum or plasma) attach toparamagnetic particles. To remove background signals associated withpotential chemical sources that may be present in the sample as well, anumber of washing steps are typically implemented into the process. Aconsequence of these washing steps, however, is that some fraction ofthe original particles will be lost for subsequent chemistry processes.

As such, there is a need for a process that allows the particlesremaining after the washing steps to be quantified in order to normalizethe luminescence signal from the patient sample. The present applicationis intended to improve upon and resolve some of these known deficienciesof the art.

SUMMARY OF THE DISCLOSURE

In accordance with one aspect of the present application, a quantitativemethod for performing an automated diagnostic assay is provided andcomprises the steps of incubating a capture reagent with astreptavidin-coated medium to form a solid phase complex; washing thesolid phase complex to remove excess capture reagent; incubating thesolid phase complex with a serum sample to form an immune complex;washing the immune complex to remove any unbound sample; incubating theimmune complex with a conjugate to create an immune-conjugate complex;washing the immune-conjugate complex to remove any unbound conjugate;introducing a substrate capable of generating a quantifiable response;and calibrating the response generated from introducing the substrate.

In accordance with yet another aspect of the present application, acontrolled process for binding fluorescent labels to particles within apatient sample is provided. In accordance with this aspect of thepresent disclosure, the process includes binding luminescent labels tothe particles, and quantifying the particles remaining after a series ofwashing steps in order to normalize a luminescence signal from thepatient sample. According to this illustrative process, the luminescentlabels are bound to the particles in proportion to a number of boundanalyte molecules.

In accordance with still another aspect of the present disclosure, aquantitative method for evaluating an allergen-specific immunoglobulin E(IgE) in a serum sample designed for use on an automated platform isprovided. In accordance with this method, biotinylated capture reagentis incubated with a streptavidin-coated solid phase to illicit adhesionof the capture reagent to the solid phase by exploitation of thebiotin-streptavidin interaction. The capture-reagent solid phase complexis then washed to remove excess biotinylation capture reagent. A serumsample is then incubated with the capture-reagent solid phase complex toillicit binding of allergen-specific IgE present in the serum to thepresented capture reagent and to create an immune complex. The immunecomplex is then washed to remove unbound IgE and then incubated withlabeled anti-IgE conjugates to illicit binding of the conjugate to theallergen-specific IgE component of the immune complex and to create animmune-conjugate complex. The immune-conjugate complex is washed toremove the unbound labeled anti-IgE and then a substrate capable ofgenerating a quantifiable response is introduced. The quantifiableresponse generated from adding the substrate is calibrated and thereported value adjusted for bead retention.

In accordance with certain aspects herein, the step of incubating thebiotinylated capture reagent with a streptavidin-coated solid phase isderived from the biotinylation of a purified allergen, protein, enzymeor antibody.

In accordance with other aspects herein, the step of incubating thebiotinylated capture reagent with a streptavidin-coated solid phase isderived from the biotinylation of an allergen extract comprised of amultiplicity of allergens.

In accordance with still other aspects herein, the step of incubatingthe biotinylated capture reagent with a streptavidin-coated solid phaseis derived from the biotinylation of an allergen extract used for invivo human diagnosis or treatment.

According to specific illustrative aspects of the present disclosure,the biotinylated capture reagent exists as an amalgam of multiplebiotinylated capture reagents of different origins including purifiedallergens, proteins, enzymes, antibodies and allergen extracts.

According to yet another specific illustrative aspect of the presentdisclosure, the streptavidin-coated solid phase is a universalfluorescent-labeled magnetic microparticle.

In accordance with certain aspects of the present disclosure, one ormore of the washing steps include washing the solid phase complexes bymagnetically sequestering the complex within a confined area of areaction cuvette.

In accordance with yet other illustrative aspects of the presentdisclosure, the step of incubating the capture reagent-solid phasecomplex with a serum sample includes incubating a capture reagent-solidphase complex that is retained in a suspension by a reaction diluentincluding a high concentration of human serum albumin (HSA).

In accordance with still another illustrative aspect of the presentdisclosure, the step of incubating the immune complex with a labeledanti-IgE conjugate comprises incubating an immune complex that isretained in a suspension by a conjugate diluent including a nominalconcentration of polyethylene glycol. According to specific aspects ofthe present teachings, the conjugate diluent is comprised of 100 ng/mLAnti-IgE-HRP, 100 μg/mL apo-HRP, 50 mM sodium phosphate, pH 6.7, 150 mMNaCl, 0.05% Tween-20, 1% BSA, 4% (w/v) PEG 6,000, 1% (v/v) ProClin 950,0.015% (v/v) Antifoam B. In accordance with yet another specific aspectof the present teachings, the conjugate diluent is comprised of 10 ng/mLanti-IgG-HRP, 10 μg/mL apo-HRP, 50 mM sodium phosphate, pH 6.7, 150 mMNaCl, 0.05% Tween-20, 1% BSA, 4% (w/v) PEG 6,000, 1% (v/v) ProClin 950,0.015% (v/v) Antifoam B.

According to certain specific aspects of the present disclosure,horseradish peroxidase (HRP) conjugated to an anti-IgE antibody can beused as an indirect label when washing the immune complex to removeunbound IgE, particularly as the reaction of PS-Atto with an HRP labeledconjugate generates sustained high-intensity luminescence for maximumdetection sensitivity in solution assays.

According to still other specific aspects of the present disclosure, theaddition of a substrate to the immune-conjugate complex comprises addingLumigen PS-Atto as the substrate capable of generating a quantifiableresponse, the quantifiable response existing as a chemiluminescentsignal generated by HRP-PS-Atto reporter system and detected by aluminometer within an optics box.

In accordance with certain aspects of the present teachings, the step ofadjusting a quantifiable response for bead retention includes the stepsof: transferring the substrate and immune-conjugate complex to an opticsbox wherein both fluorescent and chemiluminescent signals arequantified; employing a ratio of initial to final fluorescence to adjustthe quantified chemiluminescent signal for bead retention; andcalibrating the adjusted chemiluminescent signal to calculate a reportedvalue. To transfer the substrate and immune-conjugate complex to theoptics box, an automated pipette arm with a reusable pipette tipaspirating the sample can be utilized. Within the optics box,fluorescence is measured to determine bead retention, and luminescenceis measured to detect the RLU signal generated by the chemistry. Themeasurements are entered into an algorithm to generate a “bead retentionadjusted RLU” that is compared the calibration curve RLU, thereafter anIgE concentration is assigned.

In accordance with still other aspects of the present disclosure, thefluorescent label exists as Alexa Fluor 594 Biocytin and the universalmagnetic microparticle exists as Thermo Scientific SA-Speed Bead, orBangs Lab BioMag Plus Streptavidin.

Still other objects and benefits of the invention will become apparentfrom the following written description along with the accompanyingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned aspects of the present invention and the manner ofobtaining them will become more apparent and the invention itself willbe better understood by reference to the following description of theembodiments of the invention taken in conjunction with the accompanyingdrawings, wherein:

FIG. 1 is a schematic illustration of a method for performing anautomated diagnostic assay in accordance with the present application;and

FIG. 2 is a top schematic view of an automated immunochemistry analyzerand reagent system in accordance with the teachings of the presentapplication.

Corresponding reference characters indicate corresponding partsthroughout the several views. Although the exemplification set outherein illustrates embodiments of the invention, in several forms, theembodiments disclosed below are not intended to be exhaustive or to beconstrued as limiting the scope of the invention to the precise formsdisclosed.

DETAILED DESCRIPTION

The embodiments of the present application described below are notintended to be exhaustive or to limit the application to the preciseforms disclosed in the following detailed description. Rather, theembodiments are chosen and described so that others skilled in the artmay appreciate and understand the principles and practices of thepresent application.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application belongs. Although any method andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present application, the specific methodsand materials are now described. Moreover, the techniques employed orcontemplated herein are standard methodologies well known to one ofordinary skill in the art and the materials, methods and examples areillustrative only and not intended to be limiting.

Before describing in detail the illustrative automated immunoanalyzersystem and method of the present disclosure, it should be understood andappreciated herein that as a way of minimizing background signals fromexcess or unbound materials, immunoassays generally require that one ormore separation phases be carried out in the reaction cuvette. Tofacilitate the separation or washing process, a variety of techniquescan be used, including, but not limited to, well coating techniques,bead coating techniques, or the use of paramagnetic particles. Each ofthese separation media are coated with a capture reagent that will bindanalyte molecules of interest in the patient's blood sample. Inaccordance with certain aspects of the present teachings, thebiotinylated capture reagent can exist as an amalgam or mixture (i.e.,capture reagents from a similar category but from different genusspecies). As those of skill in the art would understand and appreciatedherein, numerous capture reagents are available and can be used inaccordance with the present teachings, including those available forlicense from the FDA, such as Mixed Vespid Venom Protein (mixed yellowjacket, yellow hornet, and white faced hornet). It should be understoodherein that the amount and volume of each of the individual capturereagents used in accordance with the present teachings depends on theirpotency (i.e. their ability to produce a detectable response).

When paramagnetic particles are used as the separation media, theparamagnetic particles are pulled to the wall of the cuvette by magnetsduring the washing process and then all of the liquid is aspirated. Asthose of skill in the art will understand and appreciate herein, duringconventional washing processes, some of the paramagnetic particles maybe aspirated along with the liquid and will therefore be lost forfurther chemistry processing. The loss of the magnetic particles becomeseven more significant if the immunoassay procedure involves several washsteps.

One of the objectives of the present teachings is to take into accountthe loss of paramagnetic particles that occur on an immunochemistryanalyzer during these washing processes. To accomplish this, inaccordance with certain aspects of the present teachings, the analytesof interest in a patient's blood sample bind to a capture reagent thathas in turn been bound to the surface of a paramagnetic particle.Luminescent labels are then bound to these analyte molecules. When aluminescing reagent or substrate is added to the cuvette it reacts withthe luminescent label to produce light that is detectable by theanalyzer's optical detector. In addition, if the paramagnetic particleshave a fluorescent label attached, fluorescently reading the contentswithin the cuvette will provide a means for determining the fraction ofthe particles that were lost during the wash steps.

According to certain aspects of the present disclosure, the automatedanalyzer utilizes common paramagnetic particles for the assays,including, but not limited to, magnetic beads or microparticles. Foreach assay onboard the analyzer, a capture reagent is incubated andbound to the universal particles in a reaction cuvette to produce anassay-specific, particle-based reagent, sometimes referring to herein asa capture-reagent solid phase complex. In accordance with certainaspects of the present disclosure, a capture reagent that can be usedfor performing a diagnostic immunoassay is comprised of Biotin-pAb orBiotin-allergens, 10 mM sodium phosphate, pH 7.4, 0.9% NaCl, 0.05%Tween-20, 1% (w/v) human serum albumin, 1% (v/v) ProClin 950, up to 5%(v/v) glycerol. In accordance with still other aspects of the presentdisclosure, another capture reagent that can be used for performing adiagnostic immunoassay is comprised of Biotin-Ags, 10 mM sodiumphosphate, pH 7.4, 0.9% (w/v) NaCl, 0.05% Tween-20, 1% (w/v) bovineserum albumin, 1% (v/v) ProClin 950, 1% protease inhibitor cocktail, 0.1mM DTT, 25% (up to 30%) (v/v) glycerol.

After undergoing a washing process, the patient sample, and optionally adiluent if needed, is added to the particles in the cuvettes andincubated. This results in the capture of specific analyte molecules inthe patient's blood sample. In accordance with one specific illustrativeaspect of the present disclosure, the reaction diluent (sample diluent)is comprised of 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.02%Tween-20, 1% (w/v) human serum albumin, 1% (v/v) human IgG, 1% (v/v)ProClin 950, 0.005% Antifoam-B v/v, 2% (w/v) PEG 6,000. In accordancewith yet another specific illustrative aspect of the present disclosure,the reaction diluent (sample diluent) is comprised of 10 mM sodiumphosphate, pH 7.4, 500 mM NaCl, 0.02% Tween-20, 25% (w/v) human serumalbumin, 1% (v/v) ProClin 950.

In accordance with these illustrative embodiments, it should beunderstood herein that the high percentage of HSA (25%) functions inpart to increase the viscosity of the reaction medium in order to retainbeads in suspension during the incubation step. In addition, high HSAalso reduces non-specific binding during this incubation, and improvesrelative light unit (RLU) linearity upon dilution of the patient sample.

Another washing process is then performed to remove any excess orunbound sample, and then a luminescent label and a conjugate is added tothe cuvette. When added to the cuvette, it can be expected that someportion of the conjugate will bind to the capture reagent/sample complexon the paramagnetic particles after an incubation period. The particlesthen undergo another wash process to remove any unbound conjugate, andthen the substrate is added to the cuvette and incubated for a shortperiod of time to allow the chemiluminescent glow reaction to reachequilibrium.

After equilibrium is reached, luminescence and fluorescence readings ofthe sample are taken. Since the paramagnetic particles are contained onthe analyzer in a common reagent vial and are maintained in a state ofuniform suspension before being pipetted into the reaction cuvettes,initial fluorescence measurements of the particles after they arepipetted into the cuvettes, when combined with the final fluorescencemeasurement for each test, can be used to determine the fraction of theinitial particles that remain in the cuvette after the immunoassayprocess. The fraction remaining is given by the following formula:

${{{Particle}\mspace{14mu} {fraction}\mspace{14mu} {remaining}} = \frac{F_{final} - F_{background}}{F_{initial} - F_{background}}},$

where F represents the corrected fluorescent signal (i.e., the measuredsignal corrected by the counting efficiency of the optical detector).Because the optical detector has a certain time resolution, as thenumber of photons detected per unit time increases, the likelihood oftwo photons arriving at the detector within that time resolution alsoincreases. Since these two photons cannot be resolved by the detector,they will count as a single photon. Thus, the detection efficiency ofthe optical detector decreases as the incident photon flux increases.

Because of the very high flux of fluorescence excitation photons whichinteract with and scatter from the container walls for the paramagneticparticles, there will be a certain number of photons that will becounted by the optical detector, even when no fluorescent material ispresent. This corrected background signal is represented byF_(background).

The use of a fluorescent measurement to determine the percentage of theinitial paramagnetic particles that remain in a reaction cuvettethroughout the immunoassay process is beneficial because the processdoes not limit system throughput, particularly because the process doesnot restrict the timing or parallel processing that can be achieved.Most conventional immunoassay analyzers, on the other hand, rely on veryreproducible processing of paramagnetic particles and samples, which doindeed restrict the timing or parallel processing that can be achieved,and as a result, also limit system throughput. While changes inprocessing efficiency over time in these conventional immunoassayanalyzers may go undetected, these changes can be detected withfluorescence detection. The teachings of the present disclosure permitthe use of parallel processing (e.g. multiple wash arms), which vary inwash efficiency due to minor mechanical alignment or fluidicdifferences. Fluorescence readings taken after each step of animmunoassay process are useful for verifying the equivalentfunctionality of the parallel processes.

An automated immunoanalyzer instrument and reagent system for performingdiagnostic assays for allergies and autoimmune diseases in accordancewith the above-described methods and techniques is now described ingreater detail. As this process is described, it should be understoodand appreciated herein that the disclosed instrumentation used toperform the assay can be configured to accept standard or universalcollection tubes so that a variety of different tests can be conductedby the system. Those of skill in the art will also understand andappreciate herein that there are many known methods for isolatingantigens, including allergens and autoimmune antigens from sourcematerials. Because these isolating methods are widely known and acceptedwithin the art, they are not discussed in detail herein, particularly asthose of skill in the art will recognize that any acceptable antigenisolation methods may be incorporated into the inventive system withoutdeparting from its spirit or scope. After the allergen or autoimmuneantigens have been isolated, they can then be conjugated with biotin tocreate biotinylated antigens or capture reagents. The biotinylatedantigens are then contacted with a streptavidin-linked solid support ormembrane. In accordance with certain aspects of the present disclosure,the biotinylated capture reagents can be derived from componentsincluding, but not necessarily limited to, purified allergens, proteins,enzymes, antibodies, DNA, nuclear extracts, cellular extracts andnon-protein antigens (e.g., drugs or materials cross-linked to aprotein).

As those of skill in the art will understand and appreciate herein,standard biotinylation processes and techniques commonly used fordiagnostic allergy immunoassays can be utilized in accordance with thepresent teachings; however the biotin/protein ratio for the reaction canbe optimized as needed to ensure optimum performance of the multiplebiotinylated reagents used in the chemistry. In accordance with certainaspects of the present teachings, a specific size linker arm of thebiotin reagent is NHS-PEG₁₂-Biotin. Moreover, for non-protein antigens,the material can be crosslinked to a biotinylated protein for coatingonto the streptavidin bead solid phase, while for autoimmune antigens,such as DNA, biotinylated dideoxynucleotides can be incorporated intothe DNA.

A schematic illustration of an automated diagnostic assay process inaccordance with certain aspects of the present disclosure is shown inFIG. 1. In accordance with this illustrative embodiment, magnetic beadsor microparticles manufactured with a streptavidin coating are mixed(incubated) with a known biotinylated allergen or autoimmune antigen(step 10). As those of skill in the art will understand and appreciateherein, the well-known affinity binding between streptavidin and biotinfacilitates antigen coating onto the surface of the beads and therebyallows for the use of a universal bead with on-board reagentpreparation. It should also be understood and appreciated herein thatthe amount of time and associated laboratory conditions required inorder to incubate the biotinylated capture reagents with thestreptavidin-coated solid phase in accordance with the present teachingsmay vary in light of the specific experiment being conducted, however,in accordance with certain aspects of the present disclosure, aparticularly useful incubation time range is from about 1 minute toabout 15 minutes, more particularly from about 5 minutes to about 10minutes and at a temperature of from about 2° C. to about 40° C., moreparticularly from about 36.8° C. to about 37.2° C.

As shown in Table 1 below, in accordance with this aspect of the presentdisclosure, the following beads may be used for the magnetic supportsdisclosed herein:

TABLE 1 Bead 1 2 3 Vendor Thermo Scientific B: Bangs Lab Pierce ProductSA-Coated BioMag Plus Streptavidin SA-Coated Magnetic Bead (Cat # BP628)Magnetic Bead Mean Diameter 1 μm ~1.5 μm 1 μm Size Distribution ±10% CV:30% (1.242 ± 0.402) NA (Within Lot CV) Biotin Binding 4 nmol Biotin >2μg Biotin (8-26 μg) 3.5 nmol Biotin Capacity (per mg (Biotin-ALP)fluorescein Bead)

While numerous processes can be utilized for mixing or incubating thebiotinylated allergen or autoimmune antigens with the streptavidincoated beads, in accordance with certain specific embodiments, theproducts are mixed in a reaction cuvette so that the allergens orantigens coat the beads due to the biotin/streptavidin interaction.According to one illustrative embodiment, 10 μL of streptavidin(SA)-coated bead is dispensed into the reaction cuvette, followed by 40μL biotinylated allergen or autoimmune antigens, which is mixed duringdispensation. The mixture is incubated for 1-15 minutes. Excessbiotinylated allergen or autoimmune antigen can then be washed off bypulling the magnetic beads to one side of the reaction cuvette andimmobilizing them while buffer is washed through the reaction cuvette(step 20). In accordance with one illustrative embodiment, the buffercan be comprised of 10 mM sodium phosphate, pH 7.4, 0.9% (w/v) NaCl,0.05% (v/v) Tween-20, 10 mg/mL HSA and 1% (v/v) ProClin 950. While thoseof skill in the art can utilize any readily available immobilizationtechniques known within the art to cause the magnetic beads to stay onone side of the reaction cuvette, in accordance with certain specificillustrative embodiments, an external magnet is used to immobilize themagnetic beads while the washing step is performed.

The streptavidin coated magnetic beads are then released from themagnetic field and allowed to move freely within the reaction cuvette. Abiological sample (serum or plasma) is then added to the reactioncuvette, followed by 40 μL reaction buffer, thereby re-suspending themagnetic beads (step 30). In addition to the biological sample, inaccordance with certain aspects of the present disclosure, a highconcentration of human serum albumin (HSA) is also used within thesuspension to promote macromolecular binding, as well as to keep themagnetic beads within the solution. Human IgG is added into the bufferto keep the reaction linear.

If the sample contains any antibodies (e.g., IgE, IgG) that are reactiveto any of the allergen or antigen bead coatings, such antibodies willbind during this sample incubation step. The sample incubation is keptat 37° C. for 40 minutes. After any antibodies within the patient samplebind with the beads, a second washing step is then performed to removeany non-bound patient sample (step 40). 150 μL of wash bufferconcentrate (50 mM sodium phosphate, pH 7.4, 4.5% (w/v) NaCl, 0.05%Tween-20, 0.05% (v/v) ProClin 950, 0.02% (v/v) Antifoam-C v/v) is addedto resuspend the bead and then the beads are pulled down with a magnetfor 1.5 min. After the solution is removed, the magnet is moved away,and 200 μL of wash buffer is added to resuspend the bead. The wash isthen repeated one more time.

After the second washing step is performed, the beads are re-suspendedin an antibody that is either specific for human immunoglobulin E (IgE)in the case of allergy assays or specific for human immunoglobulin G, Mor A (IgG/M/A) in the case of autoimmune assays. In accordance withcertain aspects of the present disclosure, the antibody is conjugated toan enzyme (such as horseradish peroxidase) to bind to any specificpatient antibodies that have been captured by the beads (step 50). Thebeads are then washed once again to remove any excess antibody (step60), and a highly sensitive light-forming reagent (e.g.,chemiluminescent substrate) is added to maximize detection sensitivity(step 70). Illustrative reagents that can be used as thechemiluminescent substrate in accordance with the teachings of thepresent disclosure include, but are not limited to, Lumigen® PS-atto,SuperSignal® ELISA Pico Chemiluminescent Substrate or SuperSignal® ELISAFemto Maximum Sensitivity Substrate. Those of skill in the art willunderstand and appreciate that numerous compounds of various structuralclasses, including xanthene dyes, aromatic amines and heterocyclicamines can be used to produce chemiluminescence under these conditions.These compounds are well-known within the patent literatures and arereadily available through many commercial venders. Some non-limitingchemiluminescent compounds include, but are not limited to, dioxetanetype molecules, luciferin, Lumigen® PS-2, Lumigen® PS-3, Lumigen® TMA-6,Lumigen® TMA-3.

Once the highly sensitive light-forming reagent is added to the reactioncuvette, light is produced (step 80). In accordance with certainembodiments, this light can be measured, by transferring the solution ina pipette tip into a reading station to read both the luminescent signaland fluorescent signal. It should be understood, however, that lightemitted in accordance with the present disclosure can be detected by anysuitable known detection means available within the art, including, butnot limited to, a luminometer, x-ray film, high speed photographic film,a CCD camera, a scintillation counter, a chemical actinometer orvisually. As those of skill in the art readily understand andappreciate, each detection means has a different spectral sensitivity,as such; the chosen detection device can be governed by several factorsincluding, the application and use, cost and convenience. Moreover, asused herein, a quantifiable or detectable response that can be measuredin accordance with the present disclosure implies that a positive samplewith allergen-specific IgE caused binding of Anti-IgE-HRP which wouldgenerate luminosity (i.e., RLU=relative light unit) upon addition of thesubstrate. In addition, it should also be understood herein that aquantifiable or detectable response can also apply to a quantifiableresponse generated by a negative sample.

In accordance with certain aspects of the present teachings, RLUgenerated by a positive/negative sample for any allergen-specific IgE(sIgE) is compared to RLU generated by a total IgE (tIgE) calibrationcurve. The calibration curve is generated by subjecting a range ofpre-diluted total IgE (tIgE) calibrators (which are generated from WHOstandards) evaluated using a Biotinylated Anti-IgE capture reagent.

To better understand the mechanical aspects of this disclosure, FIG. 2illustrates an automated immunochemistry analyzer and reagent system 100that can be used to quantify and normalize the luminescence signal of ananalyte sample in accordance with the teachings of the presentdisclosure. According to this illustrative aspect, the automatedimmunochemistry analyzer 100 begins by first dispensing fluorescentlylabelled paramagnetic particles, or fluo-beads, into a cuvette locatedwithin the reaction rotor 106. In accordance with one embodiment herein,an exemplary Fluo-Bead includes Fluo-Bead (SA-Speed Bead, Atto 590labeled), 1 mg/mL.

The fluo-beads may initially be located in the vortexer 102 and betransferred to the reaction rotor 106 by the R1 pipettor 104. The R1pipettor 104 can aspirate a desired quantity of the fluo-bead mixtureand transfer the aspirated quantity to the reaction rotor 106 where itis injected into the cuvette of the reaction rotor 106. Following theinjection into the cuvette, the optics pipettor 108 may aspirate a testsample from the cuvette of the reaction rotor 106 and transfer the testsample to the optics box 110. Once the sample is disposed within theoptics box 110, fluorescence and luminescence measurements can berecorded. The initial recording of the fluorescence and luminescencesignal can be used as a baseline measurement for the fluorescence signalthat can correspond to the initial concentration of fluo-beads in asample. After recording the measurements, the multi rinse pipettor 112can rinse the cuvettes using a wash buffer.

Next, fluo-beads may be transferred from the vortexer 102 to a cuvettein the reaction rotor 106 via the R1 pipettor 104. Then, the R1 pipettor104 may aspirate a capture reagent from the reagent rotor 114 and injectthe capture reagent into the cuvette located in the reaction rotor 106.After an incubation period, the single rinse pipettor 116 may inject arinse buffer to resuspend the fluo-bead. A substantial amount of thesuspended fluo-bead may then be localized by magnets within the reactionrotor 106 over a period of time. After the magnets have substantiallylocalized the fluo-beads within the cuvette, the multi rinse pipettor112 may aspirate and dispose of a portion of the rinse buffer, leaving aportion of the fluo-beads localized within the cuvette. The multi rinsepipettor 112 may proceed to inject a wash buffer into the cuvette of thereaction rotor 106, resuspending the fluo-beads. The fluo-beads mayagain be localized by the magnets within the reaction rotor 106 to befollowed by the multi rinse pipettor 112 aspirating and discarding aportion of the sample that was not localized from the cuvette in thereaction rotor 106.

A patient sample may be contained in a sample tube on in the samplerotor 118. The patient sample may further be partially diluted with asample diluent. At this point, the sample pipettor 120 may aspirate aportion of the patient sample and inject the patient sample into thecuvette of the reaction rotor 106 to resuspend the fluo-beads. Thecuvette containing the patient sample within the reaction rotor 106 maythen incubate at a specific temperature, for a specific amount of time.After incubation, the single rinse pipettor 116 may inject the rinsebuffer to again resuspend the fluo-beads. Another localization processis performed by the reaction rotor 106 by allowing the fluo-beads tosubstantially collect within the cuvette near the magnets in thereaction rotor 106. After the localization of the fluo-beads, the multirinse pipettor 112 may aspirate and discard a portion of the fluidwithin the cuvette of the reaction rotor 106 that was not localizedduring the localization process.

A couple of rinse cycles may then be performed on the sample within thecuvette of the reaction rotor 106. The rinse cycle may comprise usingthe multi rinse pipettor 112 to inject a wash buffer into the cuvette toresuspend the fluo-beads. Another localization step may allow thefluo-beads to collect within the cuvette by the magnets within thereaction rotor 106. After a period allowing for adequate localization ofthe fluo-beads, the multi rinse pipettor 112 may aspirate andunintentionally discard a portion of the sample, leaving a portion ofthe fluo-beads within the cuvette of the reaction rotor 106. Anotherrinse cycle may then occur by using the multi rinse pipettor 112 toagain inject wash buffer into the cuvette and allow the fluo-beads toresuspend. Another fluo-bead localization process may utilize themagnets within the reaction rotor 106 to localize the fluo-beads fromthe rest of the sample. Finally, the multi rinse pipettor 112 mayaspirate a portion of the sample that was not localized by thelocalization process.

At this point, the R2 pipettor 122 may aspirate a conjugate contained ina conjugate cuvette within the reagent rotor 114. The R2 pipettor 122may then inject the previously aspirated conjugate into the cuvette ofthe reaction rotor 106. After incubating the cuvette under controlledtime and temperature in the reaction rotor 106, the single rinsepipettor 116 may inject a rinse buffer into the cuvette in the reactionrotor 106. Another fluo-bead localization cycle may be performed byallowing magnets within the reaction rotor 106 to substantially localizethe fluo-beads within the cuvette. The multi rinse pipettor 112 mayaspirate and discard a portion of the sample within the cuvette that hasnot been localized during the localization cycle.

Two more rinse cycles may be performed on the sample within the cuvetteof the reaction rotor 106. The multi rinse pipettor 112 may inject awash buffer to resuspend the fluo-beads within the cuvette. Anotherfluo-bead localization cycle may localize the fluo-beads by locating thecuvette within close proximity to the magnets in the reaction rotor 106over an adequate period of time. After the localization cycle, the multirinse pipettor 112 may aspirate and discard a portion of the sample thatwas not localized during the localization cycle. A second wash cycle maythen occur by using the multi rinse pipettor 112 to inject the washbuffer to resuspend the fluo-beads. Another localization cycle mayutilize the magnets within the reaction rotor 106 to localize thefluo-beads within the cuvette. After the localization process, the multirinse pipettor 112 may again aspirate and discard a portion of thesample that was not localized during the localization cycle.

At this point, the R2 pipettor 122 may aspirate a portion of conjugatefrom the reagent rotor 114 and inject the conjugate into the mixedsubstrate container 124 creating a mixed substrate sample. The R2pipettor may then aspirate the mixed substrate sample from the mixedsubstrate container 124 and inject the mixed substrate sample into thecuvette of the reaction rotor 106, resuspending the fluo-bead with themixed substrate sample. The sample in the cuvette of the reaction rotor106 may then be aspirated by the optics pipettor 108 and placed in theoptics box 110. After the optics box makes fluorescence and luminescenceoptical observations, the sample is discarded and the multi rinsepipettor rinses the cuvettes of the reaction rotor 106 in preparationfor the next test.

Advantages and improvements of the processes, methods of the presentdisclosure are demonstrated in the following examples. These examplesare illustrative only and are not intended to limit or preclude otherembodiments of the present disclosure.

EXAMPLE 1 Biotinylation of Anti-Human IgE or Allergen Extracts

2 μL of NHS-PEG12-Biotin (Pierce) 250 mM in DMSO is added to 1 mL ofaffinity purified anti-human IgE (ImmunoReagents) 5.0 mg/mL in PhosphateBuffered Saline (PBS). Or, 1.6 μL of NHS-PEG12-Biotin (Pierce) 250 mM inDMSO is added to 1 mL of allergen extracts 1.0 mg/mL in PhosphateBuffered Saline (PBS).

The reagent solution is mixed and placed on ice for 2 hours. Free biotinreagent is separated from the biotinylated antibody by dialysis againsttwo changes of PBS (volume ratio of antibody to buffer—1:100) at 2-8° C.for 4 hours and overnight.

EXAMPLE 2 Preparation of Fluo-Bead

5 μL of Biotin-Fluo (Alexa Fluor 594 Biocytin, Sodium Salt, LifeTechnologies) 1 mM in ddH₂O is added into 45 mL of PBSTHP Buffer (10 mMsodium phosphate, pH 7.4, 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20, 10mg/mL HSA, 1% (v/v) ProClin 950). Mix well.

5 mL of SA-Speed Bead (Sera-mag Speedbeads Streptavidin-Coated MagneticParticles, Thermo) 10 mg/mL is added into the Biotin-Fluo solution andmixed well.

EXAMPLE 3 Assay for Specific IgE Levels to Allergens

10 μL Fluo-Bead (Fluorescence labeled para-magnetic microparticles) atbead concentration 1 mg/mL is dispensed into the reaction cuvette; 40 μLof biotin-allergen (e.g., Egg white, Milk, Peanut, etc) orbiotin-anti-IgE antibody, is dispensed and mixed into the Fluo-Bead, andincubated for 1-10 min at 37° C. After washing, allergen- oranti-IgE-coated beads are resuspended in 40 μL of reaction buffer. Serumsamples obtained from atopic and non-atopic individuals are assayedagainst allergens. A 10 μL sample was added to 40 μL of suspendedallergen-coated beads in reaction cuvette. For the six point standardcurve, 10 μL of serum standards (secondary standards calibrated againstthe WHO IgE Standard 75/502) are each added to 40 μL of anti-IgE-coatedbeads in a reaction cuvette. While various different labeled anti-IgEconjugates can be utilized in accordance with the present teachings, inaccordance with certain teachings, the following anti-IgE conjugates areutilized: for allergy assays—Anti-IgE-HRP; for autoimmuneassays—Anti-IgA-HRP, Anti-IgG-HRP and Anti-IgM-HRP; forECP—Anti-ECP-HRP; and for Tryptase—Anti-Tryptase-HRP. Moreover, as usedherein, each conjugate has an optimized HRP incorporation ratio for usein the chemistry. In accordance with certain aspects of the presentteachings, the rage of HRP incorporation ratio used for the listedconjugates is between about 1.2 and about 5.4. In addition, the presentteachings also contemplate the incorporation of other types ofconjugate-reporter systems including, but not limited to: alkalinephosphatase conjugate and b-galactosidase conjugate.

The solutions are mixed and incubated for 40 min at 37° C. Afterwashing, beads are resuspended in 50 μL of anti-human IgE-HRP conjugate,and incubated at 37° C. for 30 min. 50 μL of PS-atto (Lumigen) is addedinto each cuvette and the beads resuspended. The bead suspension istransferred into a pipette tip and read in the optic box for bothfluorescence and luminescence signal. The standard curve was determinedusing a four parameter logistic function equation and levels of specificIgE to allergens interpolated from the standard curve.

A list of illustrative reagents and components that may be used inaccordance with the present teachings include, but are not necessarilylimited to: Bead: Fluo-Bead (SA-Speed Bead, Atto 590 labeled), 1 mg/mL;Capture Reacient Diluent: IgE:10 mM sodium phosphate, pH 7.4, 0.9% (w/v)NaCl, 0.05% Tween-20, 1% (w/v) human serum albumin, 1% (v/v) ProClin950, up to 5% (v/v) glycerol; ANA: 10 mM sodium phosphate, pH 7.4, 0.9%(w/v) NaCl, 0.05% Tween-20, 1% (w/v) bovine serum albumin, 1% Proteaseinhibitor cocktail, 0.1 mM DTT, 1% (v/v) ProClin 950, 25% (up to 30%)(v/v) glycerol; Wash Buffer Concentrate (5×): 50 mM sodium phosphate, pH7.4, 4.5% (w/v) NaCl, 0.05% Tween-20, 0.05% (v/v) ProClin 950, 0.02%(v/v) Antifoam-C v/v; Reagent Diluent (Reaction Diluent & SampleDiluent) IgE—10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.02%Tween-20, 1% (w/v) human serum albumin, 1% (v/v) human IgG, 1% (v/v)ProClin 950, 0.005% Antifoam-B v/v, 2% (w/v) PEG 6,000; ANA—10 mM sodiumphosphate, pH 7.4, 500 mM NaCl, 0.02% Tween-20, 25% (w/v) human serumalbumin, 1% (v/v) ProClin 950; Calibrator & Control: Calibrator. Patientsample diluted into Sample Diluent; Control: Patient Sample pool;Coniudate: Conjugate Diluent: 50 mM sodium phosphate, pH 6.7, 150 mMNaCl, 0.05% Tween-20, 1% BSA, 5% (w/v) PEG 6,000, 1% (v/v) ProClin 950;IgE :100 ng/mL anti-IgE-HRP, 100 μg/mL apo-HRP, in diluent, 0.015%Antifoam-B v/v; and Substrate: PS-atto A & B, 0.01% Antifoam-B v/v.

While an exemplary embodiment incorporating the principles of thepresent application has been disclosed hereinabove, the presentapplication is not limited to the disclosed embodiments. Instead, thisapplication is intended to cover any variations, uses, or adaptations ofthe application using its general principles. Further, this applicationis intended to cover such departures from the present disclosure as comewithin known or customary practice in the art to which this presentapplication pertains and which fall within the limits of the appendedclaims.

The terminology used herein is for the purpose of describing particularillustrative embodiments only and is not intended to be limiting. Asused herein, the singular forms “a”, “an” and “the” may be intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. The terms “comprises,” “comprising,” “including,” and“having,” are inclusive and therefore specify the presence of statedfeatures, integers, steps, operations, elements, and/or components, butdo not preclude the presence or addition of one or more other features,integers, steps, operations, elements, components, and/or groupsthereof. The method steps, processes, and operations described hereinare not to be construed as necessarily requiring their performance inthe particular order discussed or illustrated, unless specificallyidentified as an order of performance. It is also to be understood thatadditional or alternative steps may be employed.

What is claimed is:
 1. A method for performing a diagnostic assay,comprising: measuring a first fluorescent signal associated with aquantity of streptavidin-conjugated fluorescent-labeled magneticmicroparticles; measuring a second fluorescent signal and achemiluminescent signal associated with a labeled immune-conjugatecomplex, wherein the a labeled immune-conjugate complex includes abiotinylated capture reagent, the streptavidin-conjugatedfluorescent-labeled magnetic microparticles, a patient antibody, and aconjugate; calculating a ratio of the first fluorescent signal to thesecond fluorescent signal to obtain a bead retention ratio; andadjusting a quantifiable response for bead retention by adjusting thechemiluminescent signal by the bead retention ratio to calculate areported value.
 2. The method of claim 1, further comprising incubatingthe biotinylated capture reagent with the quantity ofstreptavidin-conjugated fluorescent-labeled magnetic microparticles toform a solid phase complex.
 3. The method of claim 2, further comprisingincubating an immune complex formed from the solid phase complex and abiological sample with the conjugate to form an immune-conjugatecomplex.
 4. The method of claim 3, wherein the biological sampleincludes the patient antibody.
 5. The method of claim 3, wherein theincubation comprises binding the patient antibody to the biotinylatedcapture reagent.
 6. The method of claim 1, wherein the biotinylatedcapture reagent includes an allergen-specific human immunoglobulin E(IgE), an autoimmune-specific human immunoglobulin G (IgG), anautoimmune-specific human immunoglobulin M (IgM), or anautoimmune-specific human immunoglobulin A (IgA).
 7. The method of claim1, wherein the biotinylated capture reagent is derived from thebiotinylation of a purified allergen, protein, enzyme, antibody, DNA,nuclear extract, cellular extract, or non-protein antigen.
 8. The methodof claim 1, wherein the biotinylated capture reagent is derived from thebiotinylation of an allergen extract comprised of a multiplicity ofallergens.
 9. The method of claim 1, wherein the biotinylated capturereagent exists as an amalgam of multiple biotinylated capture reagentsselected from purified allergens, proteins, enzymes, antibodies, andallergen extracts.
 10. The method of claim 2, wherein the step ofincubating the biotinylated capture reagent with the quantity ofstreptavidin-conjugated fluorescent-labeled magnetic microparticlesincludes a reaction diluent including human serum albumin (HSA).
 11. Themethod of claim 3, wherein the step of incubating the immune complexwith a conjugate comprises incubating with a conjugate diluent includingpolyethylene glycol.
 12. The method of claim 1, wherein the conjugateincludes horseradish peroxidase (HRP).
 13. The method of claim 12,wherein the conjugate further includes a label.
 14. The method of claim1, further comprising transferring the labeled immune-conjugate complexto an optics box, wherein the second fluorescent signal and thechemiluminescent signal are quantified.
 15. The method of claim 14,wherein the step of transferring the labeled immune-conjugate complex toan optics box comprises using an automated pipette arm with a reusablepipette tip.
 16. The method of claim 1, further comprising: measuringfluorescence within the optics box to determine bead retention; andmeasuring chemiluminescence within the optics box to detect a generatedrelative light unit signal.
 17. The method of claim 16, furthercomprising entering the fluorescence and chemiluminescence measurementsinto an algorithm to generate a bead retention adjusted relative lightunit signal.
 18. The method of claim 17, further comprising comparingthe generated bead retention adjusted relative light unit signal to acalibration curve relative light unit signal.
 19. The method of claim 1,wherein the patient antibody is from a serum sample.
 20. The method ofclaim 1, wherein the patient antibody is from a plasma sample.